STRUCTURAL ANALYSIS OF A EUKARYOTIC CENTROMERE

真核生物染色体的着丝粒是染色体沿着 在染色体运动中起基本作用的染色质纤维。 从酵母中分离的着丝粒DNA序列 酿酒酵母，使外源DNA引入酵母的功能， 在细胞分裂过程中的普通酵母染色体。 我们将研究如何 着丝粒DNA序列与细胞中的染色质成分相互作用 核，包括组蛋白和非组蛋白蛋白。 我们已经能够 从酵母细胞中选择性切除着丝粒并亲和纯化 蛋白质-DNA复合物。 这种方法提供了一个独特的机会， 来鉴定体内与着丝粒DNA结合的蛋白质。我们会研究如何 动粒成分组装到DNA上，当 复合体附着在有丝分裂器上。 有条件地 通过从相邻启动子转录来调节着丝粒功能 允许精确的遗传控制染色体的运动在细胞 周期 我们将研究在细胞周期中， 有丝分裂力以及细胞如何监控染色体的保真度 种族隔离 条件性着丝粒和选择性点突变 着丝粒DNA的功能丧失是无法区分的，但 在失活机制上有所不同。 我们可以区分 蛋白质-蛋白质突变导致的着丝粒处的蛋白质结合 着丝粒功能所需的相互作用，并将识别突变体 蛋白质与着丝粒DNA的结合。 反式作用的鉴定 染色体运动所需的因素将使我们能够确定 这些因子与着丝粒DNA相互作用， 负责染色体运动的功能单位。 我们将确定 紧密着丝粒连锁基因SP 015在纺锤体中作用 形态发生 SP 015是一类新的GTP结合蛋白的成员， 是基于微管的机械化学酶。 知识 控制着丝粒和纺锤体组织的因素可能 提供了控制细胞的分子机制的基本原理 师.

Centromeres of eukaryotic chromosomes are specific regions along the chromatin fiber that play a fundamental role in chromosome movement. Centromere DNA sequences isolated from the yeast, Saccharomyces cerevisiae, enable foreign DNA introduced into yeast to function as ordinary yeast chromosomes during cell division. We will examine how the centromere DNA sequence interacts with chromatin components in the cell nucleus, including histone and non-histone proteins. We have been able to selectively excise the centromere from yeast cells and affinity purify the protein-DNA complex. This methodology provides a unique opportunity to identify proteins bound to centromere DNA in vivo. we will examine how the kinetochore components are assembled onto the DNA, and when the complex attaches to the mitotic apparatus. The ability to conditionally regulate centromere function by transcription from an adjacent promoter allows the precise genetic control of chromosome movement during the cell cycle. We will study when in the cell cycle chromosomes are subject to mitotic forces and how the cell monitors the fidelity of chromosome segregation. The conditional centromere and selected point mutations in centromere DNA are indistinguishable in their loss of function, but differ in the mechanism of inactivation. We can discriminate loss of protein binding at the centromere from mutations in protein-protein interactions required for centromere function, and will identify mutants in protein binding to centromere DNA. The identification of trans-acting factors required for chromosome movement will enable us to determine how these factors interact with the centromere DNA to give rise to a functional unit responsible for chromosome locomotion. We will determine the role of a tightly centromere-linked gene, SP015 in spindle morphogenesis. SP015 is a member of a new class of GTP-binding proteins, that are microtubule-based mechanochemical enzymes. Knowledge of the factors that control the organization of the centromere and spindle may provide fundamental principles governing the molecular mechanism of cell division.